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Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes.

ABSTRACT: Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous and cytokine-induced activation of STAT1 and STAT3 is impaired by FGF, as demonstrated by imaging of active STAT nuclear translocation and analyses of STAT activatory phosphorylation and transcriptional activation. Further, we demonstrate that FGF induces expression of CIS, SOCS1 and SOCS3 inhibitors of gp130, a common receptor for the IL6-family of cytokines. Since cytokine-gp130 signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth-inhibitory effect of FGFR3 in cartilage.

SUBMITTER: Krejci P

PROVIDER: S-EPMC2655766 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes.

Krejci Pavel P Prochazkova Jirina J Bryja Vitezslav V Jelinkova Petra P Pejchalova Katerina K Kozubik Alois A Thompson Leslie Michels LM Wilcox William R WR

Cellular signalling 20081012 1

Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous an ...[more]

PMID: 18950705

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Project description:Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.

Project description:Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-?B, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.

Project description:After exposure to human recombinant interleukin-1 (hrIL-1), chondrocytes increase their synthesis of prostaglandin E2 (PGE2) and neutral metalloproteinases (NMPs). This response, known as chondrocyte activation, is also elicited by partially purified preparations of rabbit synovial IL-1, known as 'chondrocyte activating factors' (CAF). CAF activates chondrocytes more strongly than does hrIL-1, probably because it contains additional growth factors. Basic fibroblast growth factor (bFGF) is one such factor, although CAF also contains others which modulate the activation of chondrocytes. Chondrocyte activation by hrIL-1 is strongly potentiated by bFGF and phorbol myristate acetate (PMA). A series of experiments was conducted to examine the possible role of protein kinase C (PKC) in mediating these effects. Inhibitors of PKC partially blocked the induction of NMPs by CAF and completely suppressed the potentiating effect of PMA. However, induction by 10 units of hrIL-1/ml and potentiation by bFGF were not affected by these inhibitors. Furthermore, cells whose PKC had been down-regulated by prolonged exposure to PMA remained responsive to IL-1. Surprisingly, inhibitors of PKC greatly increased the amounts of NMPs produced in response to a low dose (1 unit/ml) of hrIL-1. These inhibitors also enhanced the synthesis of PGE2 by cells responding to 1 and 10 units of hrIL-1/ml. Phosphorylation of the 80 kDa substrate for PKC was augmented by PMA and CAF, but not by hrIL-1 or bFGF. Moreover, Western-blotting techniques, which confirmed translocation of PKC in response to PMA and CAF, did not detect translocation in cells treated with hrIL-1 or bFGF. Western blotting also demonstrated the presence of PKC isoenzyme type III (alpha), but not types I (gamma) or II (beta). These data argue that PKC does not mediate the effects of hrIL-1 or bFGF in chondrocytes. However, CAF contains additional substances which activate this enzyme and whose effects may in part be mediated by PKC.

Dataset Information

Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes.

Publications

Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes.

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